: The overall goal of this project is to characterize the regulation of guanylyl cyclases in the olfactory system. Guanylyl cyclase exists as two fundamentally different enzymes: particulate guanylyl cyclase (the membrane-associated form) contains a single transmembrane spanning domain, an extracellular domain, which serves for ligand binding, and internally a catalytic domain and kinase-like regulatory domain. Soluble guanylyl cyclases are cytosolic heterodimeric here containing enzymes activated by a newly recognized class of diffusible gaseous messengers, nitric oxide and carbon monoxide. Preliminary evidence demonstrates that both forms of guanylyl cyclase are activated by odorants, making the olfactory system an interesting model. However, odorants induce a slower, sustained rise in cGMP due to the activation of guanylyl cyclase. This is in contrast to the odorant-induced cAMP and inositol 1,4,5-trisphosphate signals, which are rapid and transient. This suggests that cGMP does not function in the initial steps of odorant perception but in desensitization or in longer term cellular responses. Studies will utilize two systems. Rat chemosensory cilia isolated by calcium shock are a somewhat simplified subcellular fraction which yet retains competence of odorant-signal pathways and expresses high levels of odor- ant-responsive particulate guanylyl cyclase activity. Primary cultures of olfactory neuroepithelium enriched in olfactory receptor neurons retain high levels of soluble guanylyl cyclase activity (the major activity) and its identified upstream activator, CO produced by here oxygenase-2. The generality of both odorant-induced responses will be determined by cGMP radioimmunoassay. The role of upstream G-protein cascades, calcium and protein kinase activity in the odorant-dependent guanylyl cyclase response will be determined. As soluble guanylyl cyclase does not contain an intrinsic ligand binding region, the activity of its immediate upstream activator CO, produced by the action of heme oxygenase-2 will also be measured. this will be done by metabolically labeling primary cultures with [14C]-glycine to metabolically label heme, the substrate for heme oxygenase-2 which yields 14CO. Lastly, the mediators of the cellular effects of cGMP action include cAMP-phosphodiesterases, cGMP-dependent protein kinases and cyclic nucleotide-gated ion channels. The modulation of the activities of these proteins in response to cGMP will be determined. Taken together, these studies will clarify the role of cGMP in olfaction.